A decrease in secretion or an alteration in the composition of lacrimal gland fluid is a primary cause of the ocular surface problems that occur in aqueous tear-deficient dry eye resulting from lacrimal gland disease, contact lens wear, LASIK surgery, and aging. Parasympathetic and sympathetic nerves are well-known stimuli of lacrimal gland secretion and the signaling pathways activated by these stimuli have been characterized. A new type of stimulus of lacrimal gland secretion, epidermal growth factor (EGF), has been identified. Based on this finding, the following working model has been proposed for the present grant: Activation of sensory nerves from the ocular surface stimulates parasymapthetic and sympathetic nerves that innervate the lacrimal gland to release their neurotransmitters. These neurotransmitters activate specific signaling pathways to stimulate the synthesis of EGF and cause its release by ectodomain shedding from the basolateral membranes. The released EGF interacts with EGF (erbB) receptors on the lacrimal gland acinar cells activating a signaling pathway that causes secretion of proteins including the shedding of EGF family members from the apical membranes. These growth factors are released into lacrimal gland fluid to protect the ocular surface. The long term goal of the experiments described in this proposal is to test this model. From the results of the proposed study, new treatments for dry eye, based on stimulating EGF-, cholinergic, and alpha1-adrenergic-dependent signaling pathways to induce secretion, could be developed. To reach this goal the following specific aims have been proposed: 1) Which EGF receptor subtypes participate in stimulation of lacrimal gland secretion?; 2) Which cellular signaling pathways does EGF activate to stimulate lacrimal gland protein secretion?; and 3) How are the expression and release of EGF, transforming growth factor (TGF) alpha, and other EGF family members regulated? Acini will be prepared from rat lacrimal glands. Immunoprecipitation, Western blot analysis, immunofluorescence microscopy, and EGF receptor deficient mice will be used to determine if EGF activates and alpha1-adrenergic agonists transactivate EGF receptors to stimulate secretion. Biochemical assays, inhibitors, and adenovirus transduction will be used to determine the cellular signaling pathways activated by EGF compared to cholinergic and alpha1-adrenergic agonists. lmmunofluorescence microscopy, Western and Northern blot analysis, and RT-PCR will be used to determine how the expression and release of EGF and its family members is regulated.